The formation of dense irreversibly sickled cells is accompanied by a number of permanent changes in the structure and function of the cell membrane. The etiology of dense cell formation is not clearly understood but it is widely agreed that it results from dehydration, a consequence of net potassium loss. One possible cause for the potassium efflux is the transient rise in intracellular calcium concentration which is known to occur during reversible sickling in vitro. The steps by which calcium activates this potassium efflux are also poorly understood. We have identified a cytoplasmic protein, named calpromotin, which participates in calcium-dependent potassium loss in red cells and perhaps in other cells. Our own studies show clearly that calpromotin participates in the process of sickle cell dehydration. Further elucidation of the components and processes involved in calcium-dependent potassium transport in erythrocytes and the specific steps involving calpromotin will be essential to our understanding of the etiology of sickle cell dehydration. From these results we have formulated the following hypotheses: 1. Sickle cell dehydration is caused in large part by a sickling-induced passive calcium influx followed by a calcium-dependent potassium efflux; and 2. the biochemical elucidation of the macromolecules involved in calcium-dependent potassium transport including calpromotin, the potassium channel and perhaps others will improve our understanding of calcium-dependent sickle cell dehydration and allow specific therapies to be developed. These hypotheses will be tested with the following Specific Aims. First transient calcium fluxes will be assessed using the ACAS argon laser cytometer for sickle cells of different densities and sickle cells which have undergone increasing times in vitro of deoxygenation/reoxygenation cycles. Whole suspensions of cells undergoing repetitive sickling will be assayed for transient calcium fluxes using a fluorometer and for potassium efflux using a potassium microelectrode. The effects of different inhibitors on transient calcium influx and calcium-dependent potassium efflux will be evaluated. Second, the structure and function of calpromotin will be elucidated by isolating the gene for the protein, characterizing its kinase activities toward the membrane and identifying its specific arrangement as an oligomer. Finally, the membrane receptor(s) for calpromotin will be isolated and studied for potassium transport function.